Specificity in the determination of antithrombin

ABSTRACT

Methods and reagents for determining antithrombin III (AT) in body fluids by adding an AT binding partner to the sample and determining the free AT binding partner.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention concerns a method for determining antithrombin III(AT) in body fluids by adding an AT binding partner to the sample anddetermining the free AT binding partner. It also concerns a reagent thatis suitable for this method.

[0003] 2. Description of Related Art

[0004] AT is a factor of the blood coagulation system which plays aregulatory role. Blood coagulation is initiated by a cascade-likeinteraction of various proteases. The last of the successive activationsteps releases thrombin, which in turn generates fibrin monomers whichassociate to form a thrombus. The most important regulator is AT, whichcan form a complex with thrombin and also with other proteases involvedin blood coagulation that blocks the active center. The AT content inthe blood of healthy humans is within a relatively narrow range. ReducedAT contents may be due to consumptive coagulopathy, a severe liverdisease or they may be hereditary. A reduced AT content is nowadaysgenerally regarded to be a risk for thrombosis. Hence in some cases theAT content is even reduced in an acute thrombosis. Therefore the ATcontent is a valuable parameter in clinical diagnostics.

[0005] Various methods are already known for detecting AT in which an ATbinding partner is added to a sample under conditions that allow aninteraction of the AT binding partner with AT present in the sample andsubsequently determining the amount of free AT partner. Suchdeterminations may, for example, be based on immunological methods oruse chromogenic substrates. In the latter case, thrombin or activatedfactor X is, for example, added to the sample which interacts with theAT present in the sample. Excess thrombin is then determined byincubation with a chromogenic substrate which forms a coloured substancedue to the action of thrombin and evaluation of the colour generationwhere the AT content is indirectly proportional to the colour formation.Methods for determining AT are described, for example, in Bergmeyer,Methods of Enzymatic Analysis, 3rd edition, “Verlag Chemie”, vol. 5, p.441-448; I. Witt, ed., “Neue Methoden der Gerinnungsanalyse mitchromogenen Substraten”, Stormorken, “Neue Methoden derGerinnungsanalyse”, page 119-121; Odegard et al., Haemostasis 7: 202-209(1978); Fareed et al., Chromogenic Peptide Substrates (eds. M. F. Scullyand V. V. Kakkar) Churchill Livingstone (1979) 183-191 and Abildgaard etal., Thromb. Res. 11, 549-553 (1977).

[0006] A disadvantage of known methods for detecting AT by addingthrombin is that a false high AT value is obtained in the presence ofinterfering factors, e.g., drugs such as hirudin that can themselvesinteract with thrombin. This disadvantage can be avoided by usingactivated factor Xa instead of thrombin. However, at present severalfactor Xa inhibitors are under development as therapeutic agents (Ostremet al., Biochemistry 37 (1998), 1053-1059; U.S. Pat. No. 5,783,421; U.S.Pat. No. 5,721,214; WO 96/40679; U.S. Pat. No. 5,693,641; WO 97/46523;JP-96-191434 etc.). When these agents come onto the market, the sameproblems will occur with a factor Xa-based detection method as with thethrombin-based test.

[0007] Hence the object of the invention was to improve known detectionmethods and to provide a method that leads to a reliable measured resulteven in the presence of interfering factors in the sample to be tested.

SUMMARY OF THE INVENTION

[0008] This object is achieved by a method for detecting antithrombinIII (AT) in a sample which may contain an interfering factor comprising:

[0009] (a) contacting the sample with a first reagent R1 containing anAT binding partner under conditions where the AT binding partneressentially does not interact with AT but interacts with the interferingfactor,

[0010] (b) adding a second reagent R2 for a first determination of thefree fraction of the AT binding partner,

[0011] (c) adding a third reagent R3 to change the conditions such thatthe AT binding partner interacts with AT and carrying out a seconddetermination of the free fraction of AT binding partner, and

[0012] (d) determining the AT content in the sample from the differencebetween the first and second determination of the free fraction of theAT binding partner.

DETAILED DESCRIPTION

[0013] The method according to the invention comprises the detection ofAT in a sample, in particular in a body fluid such as blood or plasma,based on determining the interaction of an AT binding partner with ATpresent in the sample wherein a first determination of the AT bindingpartner is carried out without AT interaction, and subsequently a seconddetermination of the AT binding partner is carried out with ATinteraction, and the AT content of the sample is determined from thedifference between the first and second determination.

[0014] The method according to the invention is based on determining thefree fraction of AT binding partner in the sample under differentconditions. A first determination of the AT binding partner is carriedout without AT interaction, i.e., under conditions where AT that ispresent essentially does not react with the AT binding partner, i.e.,there is no interaction or only to an extent that does not substantiallyimpair the determination due to the fact that AT is, for example, notpresent in an active form. Subsequently the conditions are changed suchthat AT present in the sample can interact with the AT binding partnerby, for example, adding a suitable reagent to set up conditions underwhich the interaction, e.g., complex formation between AT and AT bindingpartner, is accelerated. The subsequent second determination of theremaining free (and active) AT binding partner allows an inference aboutthe AT content of the sample.

[0015] The free AT binding partner can basically be determined by anymethod. Chromogenic determinations of activity are preferred in which,for example, the proteolytic activity of AT binding partners such asthrombin or factor Xa is determined, or an immunological determinationis carried out in which, for example, antibodies are used which arespecifically directed towards an AT binding partner that is notcomplexed (with AT) and which do not interfere with the subsequentcomplex formation between AT and AT binding partners.

[0016] A particularly preferred embodiment of the method according tothe invention consists of determining the proteolytic activity of an ATbinding partner. In this method the proteolytic activity of the ATbinding partner remaining after reaction with an interfering factor isdetermined under conditions where AT present in the sample cannot reactor can only slightly react with the AT binding partner. Subsequently theformation of the complex between AT and AT binding partner isaccelerated by, for example, activating the AT. Complexes are formed inthis process from activated AT and the binding partner. Such a complexedbinding partner has essentially no more proteolytic activity.Subsequently the activity of the binding partner is again determined.The difference between the first and the second activity corresponds tothe amount of AT in the sample.

[0017] The AT binding partner is a detectable substance and preferably asubstance with protease activity that can form a complex with AT whichpreferably results in its inhibition. Examples of suitable AT bindingpartners are thrombin and factor Xa. Thrombin is particularly preferablyused.

[0018] The AT binding partner is preferably detected by means of achromogenic substrate which forms a colour due to the action of the ATbinding partner and measurement of the resulting colour. Examples ofpreferred substrates are peptidic substrates, for example, the thrombinsubstrate Tos-Gly-Pro-Arg-p-nitroaniline (CHROMOZYM TH, Pentapharm AG,Switzerland) which is converted by thrombin to Tos-Gly-Pro-Arg-OH andp-nitroaniline. However, other substrates that are accepted bycorresponding AT binding partners are of course also suitable.

[0019] In contrast to methods of the prior art, a first determination ofthe activity of the AT binding partner occurs in the method according tothe invention under conditions where AT present in the sample cannot, orcan only to a slight extent, complex the binding partner and inhibit itsactivity. Hence it is expedient to carry out the first determination inthe absence of substances such as heparin which accelerate complexformation between AT and AT binding partner. Antagonists for theaccelerator such as heparin antagonists, e.g., polybrene, can beoptionally added in small amounts. The addition of heparin antagonistsis especially expedient when a patient has been previously treated withheparin such that a (low) heparin concentration present in the sampleleads to an undesired acceleration of the complex formation between ATand AT binding partner. The addition of antagonists can at leastpartially prevent this undesired acceleration.

[0020] After the first determination of the free AT binding partner,another reagent is preferably added to the reaction mixture whichcontains an accelerator of complex formation such as heparin.Subsequently a second activity is determined under conditions where ATpresent in the sample can complex the AT binding partner. The AT contentin the sample can be determined from the difference between the firstand the second determination. The measured signal is inverselyproportional to the AT concentration in the sample. If required, thethird reagent can also contain additional AT binding partners. Inaddition, additional substrate can be pipetted in another step if toomuch substrate has already been consumed in the first determination.

[0021] The AT binding partner is determined by methods that arebasically known, for example, as described in the Antithrombin III testfrom Roche Diagnostics GmbH, Mannheim, Germany. The determination can,for example, comprise a kinetic test or a two-point determination.

[0022] The invention also concerns a reagent kit for the quantitativedetection of AT in a sample comprising:

[0023] (a) a first reagent R1 containing an AT binding partner,

[0024] (b) a second reagent R2 to determine the free AT binding partner,and

[0025] (c) a third reagent R3 containing an accelerator for theinteraction between AT and AT binding partner where the third reagent R3is separate from the first reagent R1.

[0026] The first reagent R1 is free of an accelerator for theinteraction, which is, for example, a complex formation between AT andAT binding partner. The first reagent can optionally also contain anantagonist for such an accelerator. The second reagent R2 fordetermining the AT binding partner can be a suitable reagent for achromogenic determination which, for example, contains a substrate forthe AT binding partner. Furthermore, the second reagent may also besuitable for an immunological determination and, for example, containantibodies against a free, unbound AT binding partner and optionallyother reagents for carrying out an immunological test, e.g., a latextest. The third reagent R3 is separate from the first reagent R1 andcontains an accelerator for the interaction, such as a complexformation, between AT and AT binding partner and is preferably heparin.

[0027] The determination can be carried out on conventional automatedanalysers such as the ROCHE/Hitachi and COBAS INTEGRA clinical chemistryanalyzers (Roche Diagnostics Corporation).

[0028] The method according to the invention is further illustrated bythe following example.

EXAMPLE

[0029] Determination of Antithrombin III in the Presence of Lepirudin

[0030] 3 μl sample solution was pipetted into a measuring cuvette. 175μl reagent R1 was added by pipette. The reagent R1 consisted of 100 mMTris-HCl, 270 mM NaCl, 12 mM EDTA, 10 g/l polyethylene glycol 6000, 1g/l bovine serum albumin, 0.5 NIH/ml bovine thrombin and a suitableamount of a fibrin polymerization inhibitor such as GPAP, pH 8.10. Itwas subsequently incubated for 5 min., and then 75 μl reagent R2(CHROMOZYM TH 1.9 mM) was added. Then the first thrombin activity wasdetermined in a kinetic test by a continuous bichromatic measurement at415 and 700 nm (primary and secondary wavelength).

[0031] Finally 175 μl reagent R3 (100 mM Tris-HCl, pH 8.1; heparin 2USP-U/ml; bovine thrombin (3.5 NIH/ml; 140 mM NaCl) was added, and thesecond thrombin activity was determined in a kinetic test by continuousmeasurement. The AT content was determined from the difference betweenthe second and first thrombin activity according to the instructions ofthe Antithrombin III kit of Roche Diagnostics GmbH, Mannheim.

[0032] This determination was carried out in the presence of differentamounts of hirudin (lepirudin, REFLUDAN, Aventis) (0, 1, 2, 4 and 8μg/ml).

[0033] The results are shown in the following table. AT concentrationdeviation (%) with the deviation (%) with lepirudin (%) found testaccording to the the test according to (μg/ml) (invention) invention theprior art 0 100 — — 1 100 0 5 2 99.9 0 10 4 102.5 3 25 8 130.0 30 52

[0034] The table shows that the lepirudin interference can be completely(deviation <5%) eliminated up to a concentration of 4 μg/ml. Thetherapeutic concentrations when administering lepirudin are usuallywithin this range, and hence the method according to the inventionreliably determines the AT content even in the presence of drugs thatmay potentially interfere.

What is claimed is:
 1. A method for detecting antithrombin III (AT) in asample that may contain an interfering factor, the method comprising:(a) contacting the sample with a first reagent R1 comprising an ATbinding partner under conditions wherein the AT binding partneressentially does not interact with AT but interacts with the interferingfactor, (b) adding a second reagent R2 for a first determination of thefree fraction of the AT binding partner, (c) adding a third reagent R3to change the conditions such that the AT binding partner interacts withAT and conducting a second determination of the free fraction of the ATbinding partner, and (d) determining the AT content in the sample fromthe difference between the first and second determinations of the freefraction of the AT binding partner.
 2. The method of claim 1 wherein theAT binding partner is thrombin.
 3. The method of claim 1 wherein the ATbinding partner is factor Xa.
 4. The method of claim 1 wherein thesecond reagent R2 comprises a chromogenic substrate.
 5. The method ofclaim 1 wherein the second reagent R2 contains an antibody fordetermining the free AT binding partner.
 6. The method of claim 1wherein the third reagent R3 contains an accelerator of the interactionbetween AT and the AT binding partner.
 7. The method of claim 6 whereinthe accelerator is heparin.
 8. The method of claim 1 wherein the firstreagent R1 further comprises an antagonist for an accelerator of theinteraction between AT and the AT binding partner.
 9. The method ofclaim 8 wherein the first reagent R1 comprises polybrene.
 10. The methodof claim 1 wherein the third reagent R3 further comprises an additionalAT binding partner.
 11. The method of claim 1 wherein the determinationof the AT binding partner comprises a kinetic determination.
 12. Amethod for the detection of antithrombin III (AT) in a sample comprisingdetermining the interaction of an AT binding partner with AT present inthe sample, wherein a first determination of the AT binding partner isconducted without AT interaction and subsequently a second determinationof the AT binding partner is conducted with AT interaction and the ATcontent of the sample is determined from the difference between thefirst and second determinations.
 13. A reagent kit for the quantitativedetection of antithrombin (AT) in a sample comprising: (a) a firstreagent R1 comprising an AT binding partner, (b) a second reagent R2 fordetermining the free AT binding partner, and (c) a third reagent R3comprising an accelerator for the interaction between AT and the ATbinding partner where the third reagent R3 is separate from the firstreagent R1.
 14. The kit of claim 13 wherein the second reagent R2 issuitable for a chromogenic determination of the AT binding partner. 15.The kit of claim 13 wherein the second reagent R2 is suitable for animmunological determination of the AT binding partner.